CARBON ISOTOPE DISCRIMINATION AND PHOTOSYNTHESIS

Annual Reviews www.annualreviews.org/aronline Annu.Rev. Plant Physiol. Plant Mol. Biol. 1989. 40:50337 Copyright©1989by AnnualReviewsInc. All rights r...
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Annual Reviews www.annualreviews.org/aronline Annu.Rev. Plant Physiol. Plant Mol. Biol. 1989. 40:50337 Copyright©1989by AnnualReviewsInc. All rights reserved

CARBON ISOTOPE DISCRIMINATION AND PHOTOSYNTHESIS G. D. Farquhar, I J. R. Ehleringer,

2 1and K. T. Hubick

~ResearchSchoolof BiologicalSciences,AustralianNationalUniversity, Canberra,

ACT 2601 Australia

2Department of Biology,Universityof Utah, Salt LakeCity, Utah 84112

CONTENTS INTRODUCTION ..................................................................................... ISOTOPE EFFECTS ........................................................................... ’ ....... ISOTOPICCOMPOSITION ANDDISCRIMINATION ....................................... Definitions.......................................................................................... IsotopicComposition of SourceAir ........................................................... "’On-line" Measurement of CarbonIsotope Discrimination.............................. THEORY OF CARBONISOTOPE DISCRIMINATION DURING PHOTOSYNTHESIS ................................................................................. C3Photosynthesis ................................................................................. C4Photosynthesis ................................................................................. C3~Intermediacy .............................................................................. Crassulacean Acid Metabolism ................................................................. AquaticPlantsandAlgae....................................................................... ENVIRONMENTAL EFFECTS ON CARBONISOTOPE DISCRIMINATION ......... Light................................................................................................. Water................................................................................................ Salinity.............................................................................................. Air Pollution....................................................................................... WATER-USE EFFICIENCY OFC3 SPECIES ............................................. : .... TranspirationEfficiency and CarbonIsotope Discrimination............................ Scalingfromthe Plant to the Canopy........................................................ CarbonIsotope Discrimination and Plant GrowthCharacteristics ..................... GeneticControlof Discrimination ............................................................ CONCLUDING REMARKS ........................................................................ APPENDIX .............................................................................................

504 504 505 505 507 508 508 509 512 514 515 516 517 518 519 520 520 520 520 522 523 524 525 525

503 1040-2519/89/0601-503502.00

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INTRODUCTION There are two naturally occurring stable isotopes of carbon, ~2Cand ~3C. Most of the carbon is 12C (98.9%), with 1.1% being 13C. The isotopes are unevenly distributed amongand within different compounds,and this isotopic distribution can reveal information about the physical, chemical, and metabolic processes involved in carbon transformations. The overall abundanceof 13Crelative to ~2Cin plant tissue is commonly less than in the carbon of atmospheric carbon dioxide, indicating that carbon isotope discrimination occurs in the incorporation of CO2into plant biomass. Becausethe isotopes are stable, the information inherent in the ratio of abundances of carbon isotopes, presented by conventionas ~3C/~2C,is invariant as long as carbon is not lost. Numerouscontributions have been made to our understanding of carbon isotope discrimination in plants since this area was extensively reviewedby O’Leary(97). Here we discuss the physical and enzymaticbases carbon isotope discrimination during photosynthesis, noting how knowledge of discrimination can be used to provide additional insight into photosynthetic metabolism and the environmental influences on that process. ISOTOPE

EFFECTS

Variation in the ~3C/~2Cratio is the consequenceof "isotope effects," which are expressed during the formation and destruction of bonds involving a carbon atom, or because of other processes that are affected by mass, such as gaseousdiffusion. Isotope effects are often classified as being either kinetic or thermodynamic, the distinction really being between nonequilibrium and equilibrium situations. One example of a kinetic effect is the difference between the binary diffusivity of 13CO2and that of ~2CO~in air. Another exampleis the difference between the kinetic constants for the reaction of ~2CO~and 13CO~with ribulose bisphosphate carboxylase-oxygenase (Rubisco). Both these examples are called "normal" kinetic effects in that the process discriminates against the heavier isotope. Thermodynamic effects represent the balance of two kinetic effects at chemical equilibrium and are therefore generally smaller than individual kinetic effects. Anexampleof a thermodynamiceffect is the unequal distribution of isotope species among phases in a system (e.g. in CO2in air versus in CO~in solution). Thermodynamic effects, like some kinetic ones, are temperature dependent. Isotope effects, denoted by a, are also called fractionation factors because they result in fractionations of isotopes. Theyare here defined (as by some, but not all chemists) as the ratio of carbon isotope ratios in reactant and product

Annual Reviews www.annualreviews.org/aronline CARBONISOTOPE DISCRIMINATION 505 Rr

Rp whereR~is the ~3C/~ZC molar ratio of reactant and Re is that of the product. Definedin this way, a kinetic isotope effect can be thought of as the ratio of the rate constants for ~2Cand ~3Ccontaining substrates, k~2 and k~3, respectively. Thus k12 O~kinetic

2.

~ 7"

A simple equilibrium isotope effect would be the ratio of the equilibrium constants for ~2Cand ~3Ccontaining compounds,K12 and K13, respectively:

Diffusional effects belong to the category of kinetic effects, and the isotope effect is the ratio of the diffusivity of the ~2Ccompound to that of the compound.The above effects are discussed more fully in Part I of the Appendix.Isotope effects mayoccur at every reaction of a sequence, but the overall isotope effect will reflect only the isotope effects at steps wherethe reaction is partially reversible or wherethere are alternative possible fates for atoms, until an irreversible step is reached (97). Kinetic isotope effects successive individual reactions are usually not additive, but the thermodynamicones are. If all reactants are consumedand converted to product in an irreversible reaction, there is no fractionation. For example, plants grownin a closed system, where all CO2was fixed, showedno isotope effect (6).

ISOTOPIC COMPOSITION AND DISCRIMINATION Definitions Farquhar &Richards (39) proposed that whole plant processes should analyzed in the same terms as chemical processes. From Equation 1 it is evident that this requires measurementsof isotopic abundanceof both source and product. For plants this meansmeasuringRa (isotopic abundancein the air) and Rp (isotopic abundancein the plant, wherethe plant can be considered the product referred to in Equation1). For numericalconvenience,instead of using the isotope effect (a = Ra/gp), Farquhar &Richards (39) proposed the use of A, the deviation of a from unity, as the measureof the carbon isotope discrimination by the plant:

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A = a- 1- Ra---

1.

4.

The absolute isotopic compositionof a sampleis not easy to measuredirectly. Rather, the mass spectrometer measures the deviation of the isotopic composition of the material from a standard, ~p = Rp - Rs = R__.e._ 1, R~ Rs whereRs is the molar abundanceratio, 13C/12C,of the standard. The reference material in determinations of carbon isotopic ratios has not normally been CO2in air but traditionally has been carbon in carbon dioxide generated from a fossil belemnite from the Pee Dee Formation, denoted PDB[for which R = 0.01124, (17)]. In this review all compositions that are denoted ~ are with respect to PDB. In contrast to 3, the discrimination, A, is independent of the isotopic composition of the standard used for measurementof Rv and Ra, and is also independent of Ra. Plants showa positive discrimination (A) against ~3C. Typically C3 plants have a discrimination of - 20 × 10-3, which is normally presented in the literature as 20%0("per nail"). Consistent with this notation, we will use %oas equivalent to 10-3. Note that "per mil" is not a unit, and is analogous to per cent; discrimination is therefore dimensionless. Equations involving the ~ notation have been madeunnecessarily complexby including the factor 1000 in the definition (i.e. ~p = (Rp/Rs - 1)°1000). Wehave opted for simplicity, but the reader should note that factors of 1000 in other treatments (including our own) should be deleted when comparing to the equations presented here. Other possible definitions of discrimination are discussed in Part III of the Appendix. The value of A as defined above is obtained from 6~ and 6p, where a and p refer to air and plant, respectively, using Equation4, and the definitions of $a and 6p (Ra/Rs - 1; Rp/Rs - 1, respectively):

-ra-rp A l+~,,"

6.

On the PDBscale, free atmospheric CO2(Ra ~ 0.01115 in 1988) currently has a deviation, 6a, of approximately -8%o, and typical C3 material (Rp 0.01093) a deviation, 6p, of -27.6%, which yields A = (--0.008 + 0.0276)/ (1 -- 0.0276) = 20.1%~.O’Leary(97) pointed out that the simultaneous of discrimination and 6 is confusing for work with plants, since the discrimination values (A) are usually positive while those of 8 are usually

Annual Reviews www.annualreviews.org/aronline CARBONISOTOPE DISCRIMINATION 507 negative whenPDBis the reference. Wherepossible, it is preferable to use molar abundanceratios (R) and compositional deviations (6) only as termediates in the calculation of final isotope effects (97). Isotopic

Composition

of Source Air

Theadvantageof reporting A is that it directly expresses the consequencesof biological processes, whereascomposition, ~p, is the result of both source isotopic compositionand carbon isotope discrimination. This distinction is particularly important in the interpretation of some growth cabinet work where the isotopic composition of CO2can be affected by mixing of CO2 derived from fossil fuel combustionwith normal atmosphericCO2.Of course, it is relevant for vegetation grownnear vents outgassing the CO2produced from burning underground coal (for which ~ = -32.5%0) (46). Of wider relevance, the distinction between/~ and A is important wheninterpreting results fromcanopies, if turbulent transfer is poor. In these conditions, there is a gradient, with height, in isotopic compositionof CO2in the air, ~a. This gradient occurs because of both canopy photosynthetic activity and soil respiration and litter decomposition. Onthe one hand, since photosynthetic processes discriminate against 13C, the remaining CO2in air should be enriched in 13C whenCO2concentration is drawndown(32, 35). On the other hand, decompositionprocesses, which release COawith an isotopic composition similar to that of the decaying vegetation, result in a muchlower lac content of the soil CO2(1, 68, 116, 122, 123, 148). Francey et al (42) reported a CO2concentration of 20 ppmlower, 1 mabove the ground, than outside the canopy in the daylight period in a dense (14 m) canopy of huon pine in Tasmania. The difference in 3, between the top and bottom of the canopy was 0.8%°. In warmand dense tropical rainforests, the COzconcentration, ca, is large near the forest floor, and 6a is small [ca = 389ppm,6a = -11.4%oat 0.5 m(133); see also (88)]. The isotopic composition, 6a, CO2concentration, ca, should be negatively related within a canopy(as in the abovereports) so that for those field-grown plants wherethe gradients of Ca are found to be small, the gradient of ~a is also likely to be small. The isotopic composition of the free atmosphere also changes, slowly becomingdepleted in t3C (41, 45, 70, 92, 108). The progressive decrease 6~ is caused by the anthropogenicburning of fossil fuels (t~ - -26%0). From 1956 to 1982, 6, has decreased from -6.7%0(at 314 ppm)to -7.9%0(at ppm) (70, 92). There is also an annual cycle of 10 ppmin c,, and 0.2%°in 3a, in the northern hemisphere, associated with seasonal changes in standing biomass; the amplitudes of changes in c,~ and 6,, are muchsmaller in the southern hemisphere(92). In major metropolitan areas, ~,~ mayvary by as muchas 2%0 both daily and annually, because of humanactivities (64, 65). Throughout

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this review when discussing studies where isotopic composition of plant material is presented without corresponding measurementsof 8a; we also provide an estimate of discrimination (A) using the assumption (for fieldgrown plants) of an atmospheric composition (Sa) of -8%°. "On-line" Measurement of Carbon Isotope Discrimination In most studies, composition of CO2from combustionof plant material (Sp) has been comparedto that of the atmospherein which the material was grown (Sa) to yield an average discrimination over the period in which the carbon was fixed. A more direct and nondestructive means of measuring short-term carbonisotope discrimination is to measurethe changesin the 13C/~2Cratio of the CO2in air as it passes a leaf within a stirred cuvette, such as those commonlyused for whole-leaf gas-exchange measurements (32, 36, 62, 125). If the reactions associated with photosynthetic CO2fixation discriminate against 13C, the remaining CO2should be enriched in 13C. Discrimination can be calculated from measurementsof the concentration (c) and the isotopic composition(~5) of the CO2of the air entering (ce and ~Se) leaving (Co and tSo) the cuvette accordingto an equation derived by Evanset al (32), 1 + 8o -- ~(8o- Be)

7.

where~ = Ce/(Ce-Co). Note that Evanset al (32) used the constant 1000in the denominatorrather than 1, because their values of ~ had also been multiplied by 1000. O’Learyet al (102) used a different "on-line" technique, where the plant was enclosed in a bell jar and allowed to deplete the CO2.The continuing isotopic enrichment of the remaining CO2was monitored and discrimination calculated from a set of differential equations. Estimates from these "on-line" methods are usually comparable to those from tissue combustion analyses (32, 62, 125). The clear advantage over tissue combustionof the "on-line" approachesis that they are nondestructive and rapid (-- 30 rain), permitting studies of isotope discrimination as function of time or of physiological and environmental conditions, The measurement of tissue is of course invaluable for longer-term integration, and for the ease with whichsmall amountsof material can be collected, stored, and subsequently analyzed. THEORY OF CARBON ISOTOPE DISCRIMINATION DURING PHOTOSYNTHESIS Carbonisotope compositionof plants was first used to indicate photosynthetic pathways in plants (2, 3, 89, 93, 106, 120, 127, 128, 130, 144, 145, 150, 151, 156, 159, 160, 163). This is because phosphoenolpyruvate (PEP)

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carboxylase, the primary carboxylating enzyme in species having a C4 metabolism,exhibits a different intrinsic kinetic isotope effect and utilizes a different species of inorganic carbon that has an isotopic composition at equilibrium different from that of Rubisco. Isotopic screening was a simple test for determining the photosynthetic pathwaywhenit was unknownfor a species. Overthe past 15-20 years, the results of such surveys have provided a broad base of the distribution of photosynthetic pathwaysamongdifferent phylogenetic groups and ecological zones (97, 99, 106). Although major photosynthetic groups could clearly be distinguished by their isotopic composition, the results of these early studies also indicated that there was substantial variation in isotopic values at both the interspecific and intraspecific levels, as well as variation associated with different environmental growth conditions and with variation in dry-matter composition. Substantial theoretical and experimentalprogress has been madeover the past ten years in understanding the biochemical, metabolic, and environmental factors contributing to the different isotopic compositions amongplants. The major isotope effects of interest are listed in Table1 and include kinetic discrimination factors associated with diffusion (denoted by a) and enzymefractionation (denoted by b), as well as equilibrium discrimination factors (denoted by Werefer to this table as we review the theory and supporting evidence. C3 Photosynthesis HIGHERPLANTSSeveral models have been developed to describe the fractionation of carbon isotopes during Ca photosynthesis (38, 69, 97, 109, 122, 149). The modelsare similar in structure, each assumingthat the major componentscontributing to the overall fractionation are the differential diffusivities of C02containing lec and ~3Cacross the stomatal pathwayand the fractionation by Rubisco.Eachof the modelssuggests additivity of fractionation factors weighted by .,the relative "limitation" or CO2partial pressure difference imposedby the step involved. Of the models, that of Farquharet al (38) has been the most extensively developedand tested. Their expression for discrimination in leaves of C3 plants in its simplest form is, A a= Pa

Pi q_ b Pi --= a + (b- a) P__L,

-Pa

Pa

8.

l)a

where a is the fractionation occurring due to diffusion in air (4.4%0, theoretical value that has not been confirmed experimentally), b is the net fractionation caused by carboxylation (mainly b3, discrimination by Rubisco; see Table 1 and also Part IV of the Appendix)and p,, and p; are the ambient and intercellular partial pressures of CO2,respectively. Equation8 is derived in Part II of the Appendix;see also reference 5.

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Table 1 Isotope effects of steps leading to CO2fixation in plants. Process

Isotope effect (,~)

Discrimination (%~)

Symbol Reference

Diffusion of CO2in air through athe stomatal pore Diffusion of CO2in air through the boundary layer to the astomata Diffusion of dissolved CO 2 through water Net Ca fixation with respect to p~ Fixation of gaseous CO2by

1.0044

4.4

a

Craig (16)

1,0029

2.9

ab

Farquhar (33)

1.0007

0.7

a~

O’Leary (98)

Rubisco from higher plants Fixation of HCO~-by PEP carboxylase

Farquhar & Richards (39) Roeske & O’Leary b(119) Guyet al (50) O’Learyet al (101) Reibach & Benedict (117) Farquhar (33)

1.027

27

b

1.030 (pH = 8)

30

b3

1.029 (pH = 8.5) 1.0020 1.0020

20 2.0 2.0

b3 b4*

-5.7

b4

-9.0 -9.0 1.1 1.1

eb

Fixation of gaseous CO 2 (in equi- 0.9943 librium with HCO~-at 25°C) by PEP carboxylase Equilibrium hydration of CO2 0.991 at 25°C 0.991 Equilibrium dissolution of CO2 1.0011 into water 1.0011 Theoreticalvalue bDatacorrectedfor dissolutionof CO~

.e,

Emrich et al (31) Mooket al (91) Mooket al (91) O’Leary (98)

The significance of Equation 8 is that when stomatal conductance is small in relation to the capacity for CO2 fixation, p; is small and A tends towards 4.4%0 (see Figure 1). Conversely, when conductance is comparatively large, pi approaches Pa and A approaches b (perhaps 27-30%0; see Appendix Part IV). Nevertheless, it is a little dangerous to take the argument further and say that when Pi and A are small, stomata are necessarily limiting photosynthesis. That conclusion would only follow if the relationship between assimilation rate, A, and p; remained linear beyond the operational point (40). There are several cases where measurements of both A and PdPa have been made in controlled conditions. Farquhar et al (35) found a significant correlation between A in dry matter and discrete measurements of Pi/P, among different species over the range of p~/pa 0.3-0.85. The best fit, taking a as 4.4%0, was observed with a value for b of 27%0. The leaf with the lowestpi/p, was from an Avicennia marina plant, which showed discrimination of 11.8%0. Such low values of A had previously been considered to be in the range of C~ plants. Downton et al (using spinach; 21) and Seemann & Critchley (using beans; 124) also observed significant correlations between A in dry matter and p~/p~, the best fit being obtained by setting b equal to 28.5%0 and 26.4%0,

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511

C3 ¯ C4 o

i~11 ._

2O

10

4 0 0

0.2

0.4

0.6

0.8

1.0

Pi/ Pa(,bar/ bar Figure 1 Carbon isotopediscrimination, A,versusthe ratio of intercellularandambient partial pressuresof CO2, P~/Pa,wl~enall are measured simultaneously in a gasexchange system(36). Theline drawnis Equation8 witha = 4,4%¢and b = 27%c. respectively. However,it should be noted that in none of the above studies was ~ directly measured.Winter(155) showedthat both A and Pi/Pa of leaves becamesmaller as Cicer arietinum plants were water stressed. Conversely, Bradford et al (9) showedthat both were greater in a tomato mutantlacking abscisic acid (ABA)than in its isogenic parent. Phenotypicreversion of A and Pi/Pa occurred when the mutant was sprayed with ABAduring its growth. Measurementsof mistletoes and their hosts (25, 30) showedinterspecific variation in both A and Pi/Po. Guyet al (52) found that increased salinity decreased A in Puccinellia and Pi/Pa as expected from theory. Overthe short term, Brugnoli et al (11) showedthat the assimilation-weightedvalue ofpi/pa and A of sugar produced by a leaf in a single day followed the predicted theoretical relationship (Equation8) with a fitted value for a of 4.1%oand for b of 24--25.5%0.The overall discrimination to starch appearedto be slightly smaller. In all of the abovecases, A, inferred from the carbon compositionof leaf material, and pi/Pa were positively correlated. The values of b that gave the best fit showedvariation, whichcould have manycauses (see Part IV of the Appcndixfor further elaboration). NONVASCULAR PLANTSSurveys of isotopic composition have been made on species of mosses, liverworts, and lichens. Isotope ratio variation in the

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range of -21.3%o to --37.5%0 (A = 13.6-30.4%o) has been reported (121, 128, 135, 136). For mosses, and some liverworts, the gametophytes are morphologically similar to higher plants but are restricted in size by their lack of vascular tissue. Their leaflike photosyntheticstructures tend to be just one cell layer thick and do not have the specialized anatomyof higher plants. They do not consistently have an epidermis with impermeablecuticle and stomata, so we might not expect to observe variation in isotope discrimination arising from short-term variation in permeability to gases as with higher plants. It is possible, however,that permeability changeswith water content. Evenif this resistance remains constant, the gradient in partial pressure across it will change if the flux changes. For example, assimilation rate may change because of differing light levels, and this should increase the gradient and decrease A (32). For other liverworts with a thicker thallus and an epidermis, there maybe pores that lead to air chambers,like stomata in higher plants, and wewouldexpect to see variation in discriminationsimilar to that in higher plants. In contrast to our explanations for variation in A in mossesand liverworts, Rundelet al (121) attributed the very negative values of/Sp in mossesin humid conditionsto a large contentof lipid in the tissue of those species [as lipids are depleted in ~3Ccomparedto other plant compounds(97)]. Teeri (135) gested that these differences mayhave arisen because of differences in the amountof carbon fixed by PEPcarboxylase, but this is unlikely to differ from that in higher plants. Among lichens, there are differences in carbon isotope discrimination that dependon the phycobionts in the symbiotic association (74, 76). Greenalgae as phycobionts are able to maintain positive photosynthetic rates whenonly misted, whereaswhencyanobacteria are the phycobionts, surface liquid water is required for photosyntheticactivity (75). This difference suggests that the CO2diffusion rate maybe limiting whencyanobacteria are the phycobionts; correspondingly, the carbon isotope discrimination by lichens with cyanobacteria is 2-4%0less than that of lichens with green algae. Anotherpossibility is that liquid water is neededfor a bicarbonate transport system, whichalso has a characteristically smaller discrimination (see the section belowon algae). further complicationis that discrimination by Rubisco, b3, is 21%oin the only cyanobacterium measured compared to 29%oin higher plants (50). A great deal more work is required before an equation like Equation 8 could be applied with confidence to lichens. C4 Photosynthesis Variation in isotopic composition amongplants with the C4 photosynthetic pathwayis less than in C3 plants, because the term b from Equation8 (largely reflecting b3, the discrimination by Rubisco, ~ 30%0)is replaced by (b4

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b3~b) which is numerically muchsmaller than b3. This is because b4 (the effective discrimination by PEPcarboxylase) is - -5.7%0(see Table 1) ~b [the proportion of the carbon fixed by PEPcarboxylation that subsequently leaks out of the bundle sheath, thereby allowing limited expressionof Rubisco discrimination (b~)] is necessarily less than unity (33). The bases for modelof discrimination are as follows: CO2diffuses through stomata to the mesophyllcells, where it dissolves (es) and is converted to HCO~(eb). At equilibrium, the heavier isotope concentrates in HCO~-comparedto gaseous CO2--i.e. the combinedterms es + eb are negative (Table 1). In turn, PEP carboxylasediscriminates against Hl3CO£----i.e.b$ is positive and normalfor a kinetic effect. Thusif the gaseousintercellular CO2is in equilibrium with HCO~-,then the net discrimination from CO2to OAAis 9.

b4 = es + eb + bf[

which is negative because of the magnitudeof eb. Various transformations then occur, dependingon Casubtype, but the net result in all cases is that CO2 is released within the bundle sheath cells and refixed by Rubisco. There is little opportunity for discriminationin the release of CO~in the bundlesheath cells because of the lack of significant biochemical branches. No further discrimination wouldoccur if the bundle sheath were gas tight (153). However, somequantities of COzand HCO~are likely to leak out of these cells and into the mesophyllcells, especially through the apoplastic portions of the bundle sheath cells, wherethey can then mix with other CO2that has diffused in through the stomata. The leak is a branch from the mainpath of carbon and allows somediscrimination by Rubisco in the bundle sheath cells (bs). Various models (18, 33, 56, 110, 117) have addressed aspects of the ~3C discrimination during C4 photosynthetic metabolism.Intrinsic to all of these modelsis the notion that variation in isotopic compositionin Ca plants is associated with leakage of C02 and/or HCO~-.The "leakiness" (~b) mayalso be regarded as a measureof the "overcycling" by PEPcarboxylase that occurs in mesophyll cells, raising the partial pressure of CO~within the bundle sheath cells (33). Farquhar (33) developedan expression for the discrimination occurring Ca photosynthesis, in which A=aPa- Pi+(b4+b3~b) Pa

__Pi =a+(b4+b3~ Pa

- a) --.Pi Pa

10.

Depending whether (b4 + b~,;b - a) is positive, zero, or negative, the dependenceof A on Pi/P,~ will be positive, zero, or negative. Experimental evidence suggests that the factor is often close to zero, with short-term

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discriminationrespondinglittle to variation inpi/Pa (32, 36, and see Figure 1). FromTable 1 it maybe seen that the zero value is obtained with ~b = 0.34. Farquhar (33) and Hattersley (56), using the Farquhar model, predicted bundle sheath "leakiness" above (k = 0.37 (the value differs from 0.34 because a smaller value was assumed for b3) should result in a positive response of A to increasing Pi/Pa. Anatomical variations between C4 types (55) maybe associated with variations in ~b. For example, Ehleringer Pearcy (29) observed that quantumyields for CO2uptake are lower for all dicots and NAD-ME (malic enzyme) C4 type grasses than for NADP-ME PCK(phosphoenolpyruvate carboxykinase) types, which have bundle sheaths with suberized lamellae (12, 57). Diminished quantum yields are to expected as a result of increased leakiness--i.e, increased overcycling within the mesophyll cells. The measureddifferences in carbon isotope discrimination by NAD-ME,NADP-ME,and PCK type Ca grasses as deduced from isotopic composition (10, 56, 150, 163) and from "on-line" measurements (32, 36) are consistent with the expectationthat leakage is greatest in the first type. Becauseth is a measureof the overcycling as a proportion of the rate of PEPcarboxylation, it is likely that A will increase wheneverRubiscoactivity is diminished more than PEPcarboxylase activity by sometreatment. Thus ~b and A depend as much on coordination of mesophyll and bundle sheath activity as on anatomicalfeatures.

C3-C4Intermediacy Monsonet al (90) measured isotopic composition of C3 -C 4 in termediate species in Flaveria and reported A values of 9.6-22.6%0.Theysuggested that the isotopic variation resulted from differences in bundle sheath leakage (according to Equation 10). While this probably accounts for some of the variation, another biochemical factor may also be important. The C3-C4 "intermediate" species appear to have glycine decarboxylase confined to the bundlesheath cells (63, 115). The effect is that CO2released by photorespiration is released and partially refixed in the bundlesheath, so that discrimination by Rubisco can occur twice (S. von Caemmerer, unpublished). The modification to b, the C3 carboxylation parameterfrom Equation8, is thus the product of the proportion, As~A,of carbon fixed twice (whereAs is the rate of assimilation in the bundle sheath, and A that by the whole leaf), and ~b, the proportion of the carbon supplied to the bundle sheath that leaks out. In the simplest form the equation becomes(G. D. Farquhar, unpublished)

l +pa chA~]Pi =a +[b(1A ]Pa + ~-~-~-~) -OIpj’jpO A=aP"-P’~-~+b( This theoretical prediction awaits experimental testing.

11.

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Acid Metabolism

The details of Crassulacean acid metabolism (CAM)that affect A have been recently reviewedby O’Leary(99). In this section, we present equations for A analogousto those discussed earlier for C3 and Ca carbon assimilation pathways. Plants in the full CAM modetake up CO2and synthesize oxaloacetate using PEP carboxylase, and the oxaloacetate (OAA)is then reduced and stored as malate (103). At dawn the plants close their stomata and decarboxylate the malate, refixing the released CO2using Rubisco. The malate that is stored at night will showthe samediscriminationas for Ca species with zero leakage (33), i.e. A=a+(b4

- a)

--.Pi Pa

12.

Winter (154) reported that nocturnal values of Pi/Pa in Kalanchoepinnata started at a Ca-like value (- 0.4) and increased with time to a C3-1ikevalue (- 0.7) before dawn. Onthis basis, we could expect instantaneous A of the carbon being fixed to have decreased from -- 0.4 to -2.7%~ as the night progressed. This is consistent with observations that A of crystalline oxalate and of carbon-4 of malic acid were near to zero (58, 100). If the stomata closed completely at dawn,the photosynthetic tissue would form a closed system and there would be no fractionation of the carbon betweenmalate and the sugar products. However,consider the case where the stomata were not closed in the light while a CAMplant was enclosed in a cuvette with no external CO2.In this case, there should be a discrimination in going from malate to the new C3 carbon because we no longer have a closed system. The discrimination is given by 4,(b3 - a), where4, is the proportion of decarboxylatedcarbon that leaks out of the leaf. Nalborczyk(94) allowed CAM plants to fix CO2only at night and found that the overall discrimination was -- 3%0.This result implies that 4’ was about 0.05--0.15. However,when CAM plants are growingin normalair, evolution of CO2in the light is usually negligible. Towardthe end of the light period, after decarboxylationof all the stored malate, there is sometimesCO2uptake [denoted phase IV by Osmond(103)] via Rubisco, and possibly involving PEPcarboxylase as well. Nalborczyket al (95) allowed plants to fix carbon only in the light and observed a discrimination of 21%~,which is what one wouldexpect with a typical C3 value of P~/Pain Equation8. Thereforein the simplest case of C4 fixation in the dark and Ca fixation in the light, the average discrimination over a 24 hr period is

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FARQUHARET AL D dt + __ A(b4 - a) ~ A(b - a) Pa dt Pa A=a+

f

~Adt f°Adt÷ f



13.

whereA is the assimilation rate, fDdt denotes the time integral in the dark, and fLdt that in the light, and b for the light period is the averageof b~ and b4 weighted by the rates of RuBPand PEPcarboxylation (if the latter occurs), respectively. Aquatic

Plants

and Algae

Carbon isotope combinations measured in aquatic plants range between -11%~and -39%0, potentially leading to the mistaken impression that both C3 and Ca photosynthetic pathwaysare present in aquatic plants (4, 22, 105, 113, 132). However,with limited exceptions (86, 147), C4 plants are known from aquatic habitats. WhenCO~fixation is via the normal C3 pathway, Equation 8 applies, but with the parameter a modified to reflect diffusion in the aqueous phase (es + al) so that A = (es + a~) p’~ -- p’-~--~ + b Pc. = e., +a~ + (b - es - a~) P--f--~, Pa P, Pa

14.

where the equivalent partial pressure of CO2at the site of carboxylation is denoted as Pc. Note that the discrimination during diffusion of CO2in water (at) is 0.7%0(98) and not 11%oas someauthors have-written. Muchof the diffusion of inorganic carbon in the aqueousenvironmentwill be as bicarbonate rather than CO2,but the discrimination here should also be small (38). Note further that the discrimination is with respect to gaseous CO2in equilibrium with the aqueous environment. However, there is a widespread mechanism(s) amongmarine and freshwater organismsfor raising the concentration of CO2at the site of carboxylation above that of the environment (5, 80). Farquhar (33) suggested that equation for C4 discrimination could be adapted to describe discrimination if the active species transported is bicarbonate as A = (es + ai) p~ - p~ + (e~ + eb +bm+ =es + at + (eo +bm+b3qb- at) P-~-~, P,

b3~b)

p--Lc 15.

is the fractionation during membrane transport. The value of bm is unknown,but it has been cautiously assumedto be small, making(e~ + eb + where bm

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517

b~n), whichis the analog for b4 from the C4 model, close to -7.9%~(33). Note that in both Equations 14 and 15 the discrimination is again expressed in relation to a gaseOussource. As with Equation 14, the discrimination in relation to dissolved COzas the source, provided it were in equilibrium with the gas phase, would be found by subtracting es and with reference to bicarbonate (again, if in equilibrium) wouldbe found by subtracting es + eb. However,it is convenient to retain the sameconvention for source carbon as used for aerial plants (i.e. gaseous COz), especially whenwe have chosen gaseous CO~as our substrate for carboxylation by Rubisco(see definition of b3 in Table 1). The latter choice is also reasonable in a mechanistic sense because the Rubisco site, with RuBPbound, probably reacts with gaseous substrates only. The effects on A of induction of active carbon accumulationwere elegantly demonstrated by Sharkey & Berry (125). The green alga Chlamydomonas reinhardtii was grownat 5%CO2and then transferred to normal air levels of CO2.Before transfer, A was 27-29%0,and after 4 hr of induction A was 4%0. Sharkey&Berry (125) discussed their results in terms of slightly simplified versions of Equations 14 and 15. Berry (5) noted that measurementsof A alone are insufficient to distinguish betweena CO2concentrating mechanism (Equation 15) and a normal C3 mechanism (Equation 14) with a large resistance to diffusion. In both cases, A is small because most of the COz reaching Rubiscois fixed. ENVIRONMENTAL DISCRIMINATION

EFFECTS

ON CARBON

ISOTOPE

Goudriaan &van Laar (47), K6rner et al (72), and Wonget al (161) amongthe first to note a strong correlation betweenthe photosynthetic rate and leaf conductance. This correlation was maintained over a wide variety of plant species and under a diversity of environmental treatments, implying some level of regulation between COzdemandby the chloroplasts and COz supply by stomatal control. If in fact there were no deviations from the slope of the photosynthesis-versus-conductance relationship and if the intercept were zero (as was the case in the original papers), then the intercellular CO2 pressure (Pi) of all plants would have been constant, dependent only photosynthetic pathway. This constancy was mistakenly suggested in at least one early review (126). Althougha numberof studies that followed showed significant tendency for photosynthesis and conductance to be correlated (161), manyof these data sets exhibited somedeviation from a linear relationship or a nonzerointercept (112, 152). It is unfortunate that in the search for general patterns the variance in Pi was, for a time, ignored. Whenit was recognized that there was a fundamentalrelationship betweenA or 6p and Pi,

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FAI~QUHARET AL

moreeffort was put into documentingand understandingthe isotopic variation at both the environmentaland genetic (intra- and interspecific) levels. In the next sections, we describe what is knownabout the relationship betweenpi(as measuredby isotope discrimination) and environmental parameters. Light While some of the first experiments reported no consistent pattern between leaf isotopic composition, ~p, and irradiance (129), later studies have indicated that ~p increased with an increase in growthirradiance. Interpretation of carbonisotope compositionof leaves experiencingdifferent light levels has been somewhatcontroversial. The controversy lies in separating the effects of light on discrimination from correlated effects on ~Sa (source air), both whichaffect leaf carbon isotopic composition. In field studies, Vogel(148) was amongthe first to describe a consistent pattern of isotopic variation in leaves under canopyconditions where light levels varied substantially. He noted that 8p within a canopy decreased by 3%0between the top (19 m) and bottom(1 m) of the canopy. He further noted that the isotopic composition soil CO2was approximately - 19%o,while that of the atmosphere was only -7%0.He attributed all of the decrease in ~5e of leaves at lower layers to a recycling of soil CO2(a lighter source CO2),although the isotopic composition of CO2within the canopy, ~Sa, was not measured. He calculated that recycled CO2accounted for 15%of the carbon incorporated in lower leaf layers--assuming that the physiological discrimination was constant. Medina & Minchin(87) pursued these observations, reporting ~;13Cgradients of 4.7 and 5.6%obetweenupper and lower canopyleaves for two different tropicalforest types. Again the decrease in dil3C of leaves at lower levels was attributed to a lighter source CO2,with the implication that as muchas 20%of the carbonfixed in lowerleaf layers wasderived fromsoil respiration. A third study by Schleser &Jayasekera (122) reports a similar pattern for fores~ beech and isolated lime trees. Again, they attributed this result to recycled soil CO2. Somerecent studies have examinedboth ~Sa and ~e- In their study in a huon pine fores’t, Franceyet al (42) observedthat ,Sp decreasedwith canopydeptti, but without ~;a decreasing in a corresponding manner, which indicates a .physiological effect. They found that leaves from lower in the canopy had greater p~ values than those from the upper canopy, suggesting, according to Equation8, a greater discrimination in lower leaves. Ehleringer et al (27, 28) observeda similar pattern with ten shrub and tree species from a subtropical monsoonforest. Leaf ~5e decreased (i.e. becamemore negative) and p~ increased as observations were madedeeper in the canopy. Furthermore, when only outer canopy leaves were measured on plants with differing degrees of canopyclosure, ~p was decreased with decreasing irradiance, consistent with the modelof increasing p~ at lower light levels. These measurementswere

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519

confirmed with gas exchangeobservations of the dependenceof Pi on irradiance. While it is undoubtedly true that a fraction of the soil CO2is incorporated within leaves at the lower canopylevel, muchof the decrease in leaf isotopic composition is likely to be associated with stomatal and photosynthetic effects. Higher Pi values in understory leaves are likely to benefit plant performance when leaves are exposed to higher irradiances during sunflecks and whenleaves are allowed to operate at higher quantum yields (71, 107). In the field, effects of irradiance on pl are difficult separate fromthose of leaf-to-air vapor pressure difference (vpd). The smaller vpd at the bottom of the canopy could also cause greater p~, and greater A (another complication is discussed after Equation A13in the Appendix). Water PHYSIOLOGICAL RESPONSE TO DROUGHT Whensoil moisture levels are decreased, a commonresponse is simultaneous decreases in photosynthesis, transpiration, and leaf conductance(40). If the "supply function" of photosynthesis (leaf conductance)decreases at a faster rate under stress than the "demandfunction" [photosynthetic dependence on Pi, sensu Farquhar & Sharkey (40)], then p~ will decrease. This effect should be measurable either an increase in 6p or correspondinglyas a decrease in A. Overthe short term whennew growthhas not occurred, the impact of stress can be detected in carbohydrate fractions within leaves (11, 163a, 81). Alternatively, the reduction in pi/Pa can be measured using the "on-line" approach (62). longer-term observations under both growth-chamberand field conditions, plants under water stress induced by low soil moisture availability produced leaves with lower Pi values as estimated by carbon isotopic composition(19, 23, 26, 39, 59-62, 131, 140, 155). Increasing the soil strength (physical resistance to root penetration), such as might occur in drier soils, induces reduction of A, as observed with reduced soil moisture levels (84). Anincrease in the leaf-to-air vapor pressure difference will also cause diminution ofpi and A in the short term (11) and long term (35, 39, 157). PHOTOSYNTHETIC PATHWAY SWITCHING In response to changes in leaf water status, a numberof species show dramatic shifts in carbon isotope composition (up to 10--15%o) associated with changes in photosynthetic metabolism. Thus uponexposure to increased drought, somespecies can shift from C3 to CAM photosynthesis (8, 54, 67, 78, 137-140, 146, 158). Correspondingly,there is an increase in ~p (decreasein A). This shift in metabolism is reversible, dependentprimarily on plant water status, and can occur in both annual and perennial leaf succulents of arid habitats. Other plants, notably "stranglers" of tropical habitats, exhibit CAM metabolismas epiphytic juveniles, but later switch to Ca metabolismwhenroots reach the soil surface (111, 134, 143).

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FARQUHARET AL

PHOTOSYNTHETIC TWIGSANDSTEMS In an interesting twist on the photosynthetic-shift theme, at least two stem succulents native to southern Africa exhibit C3 metabolism in the leaves (which are shed early in the drought period) and CAMin the stems (77, 142). In recent studies on green-twig plants from arid lands of North America, high rates of photosynthesis have been observedin twig tissues that are comparableto those observed in leaves (13, 24, 104, 131). Unlikethe previous example,the twigs of these species all have C3 photosynthesis. In all such species examinedto date, Pi values as measuredby gas exchange techniques are lower in twig than leaf tissues, leading to a significant difference in carbon isotopic compositionof the two tissue types. Thus, in these cases, the decrease in h of the twigs is associated with increased diffusional constraints rather than with a changein metabolic pathwayas described in the previous section. Salinity In nonhalophytic species, increased salinity has numerousmetabolic effects (48). Stomatal closure is typically associated with increased salinity (20, 79, 124). Thus it should not be surprising to note that in those species A decreasedwith increasing salinity, indicating a decrease in pi with increasing stress (124). Whatis perhaps more intriguing is that halophytic species also exhibit a similar pattern whetherin field or laboratory conditions (35, 51-53, 96, 163a). Air Pollution A long-term consequenceof exposure to air pollutants (e.g. ozone, sulfur dioxide) at the leaf level is normallya decrease in both leaf conductanceand photosynthesis (118). It is not clear, however,whether this decrease in gas exchangerepresents overall decline in metabolic activity or an increased diffusion limitation imposedby stomata. In each of the limited numberof studies available that examine carbon isotope discrimination by leaves of plants exposed to pollutants, exposed plants exhibited lower A values, suggesting lower Pi (43, 49, 81). Changesin isotopic composition of leaf tissues from these studies of 1%oor greater were common even under modest exposures to air pollution. Underlong-term, chronic exposure to air pollutants, clear differences exist in the carbonisotope ratios of the woodof annual growthrings that are consistent with short-term,-leaf-level observations (43, 44, 81). WATER-USE

EFFICIENCY

OF C3 SPECIES

Transpiration Efficiency and Carbon Isotope Discrimination Measurements of A in C3 species mayusefully contribute to the selection for transpiration efficiency--i.e, the amountof carbon biomassproducedper unit water transpired by the crop.

Annual Reviews www.annualreviews.org/aronline CARBONISOTOPE DISCRIMINATION 521 The instantaneous ratio of CO2assimilation rate of a leaf, A, to its transpiration rate, E, is given approximatelyby

where~, is the water vapor pressure difference betweenthe intercellular spaces and the atmosphere. The factor 1.6 arises because the binary diffusivity of water vapor and air is 1.6-fold greater than that of COzand air. Equation16 maybe rewritten as

A E

1.6~,

17.



to emphasizethat a smaller value ofpi/p,~ is equivalent to an increase in A/E, for a constant water vapor pressure difference, u. Thusselecting for lower Pi/P,~ should be, to a first approximation,a screen for greater A/E, which, in turn, is a componentof transpiration efficiency. FromEquation 8, A maybe used as a surrogate measureof Pi/P,, in Ca plants. In all of the experiments relating gas exchangeproperties and short- and long-term discrimination (see the section above on C3 photosynthesis) and where vapor pressure difference, u, was maintained constant, the ratio of assimilation and transpiration rates, A/E, was negatively related to A, as expected from Equation 17. However,during whole-plant growth, losses of carbon and water occur that are not included in Equation 17. A proportion, ~bc, of the carbonfixed via the stomataduring the day is lost fromthe shoot at night or from nonphotosyntheticorgans such as the roots, during both the day and night. Further, somewater is lost from the plant independently of CO2 uptake. The stomata maynot be completely closed at night, cuticular water loss occurs, and there is unavoidable evaporative loss from the pots in whole-plant experiments. If this "unproductive" water loss is a proportion, 4’w, of "productive" water loss, Equation17 maybe modifiedto describe the molar ratio, W, of carbon gain by a plant to water loss p,~(l- Pi](1-~b¢)p,,/ W =

1.6~’(1 + ~w)

,

18.

which, when combinedwith Equation 8, predicts a negative linear dependence of Won A (38, 60). By substitution, Equation 18 can be rewritten

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FARQUHARET AL Pa

W=

- a - a)(1 _ (kc) b - a 1.6v(1

+ (k,~)

19.

where d is a correction related to assimilation rate (see Part III of the Appendix). The data from pot experiments using a combination of watering treatments and genotypes fit the theory reasonably well for a numberof species--wheat (39, 84), peanuts (61, 62, 162), cotton (59), tomato(83), barley (60). Wesuggest that future studies will provide better understanding of the relationships betweenWand A whenaccount is taken of environmental and genetic effects on (kc and (kw. Scaling from the Plant to the Canopy Water-useefficiency is difficult to measurein the field. Therehave, however, been a few attempts to .relate it to A, or at least to relate yield under water-limited conditions to A. Wrightet al (162) measuredtotal above-ground biornass yield and water use of eight peanut genotypes receiving adequate water (under a rain-excluding shelter). They obtained a negative relationship between Wand leaf A. There are several reasons whythe negative relationship betweenWand A, given by Equation 19, might work well for individual plants in pots, or even for small plots in the field, but becomeinconsistent over larger areas. The uncontrolled loss of water is not an independent, fixed proportion ((kw) transpiration because, for example, soil evaporation tends to be negatively related to leaf area development.If v fluctuates, then those genotypes that might grow more when v is small will obtain a greater Wfor the same A. Equation 19 also contains a simplification that becomesmore problematic with increase of scale. The equation is written as if the vapor pressure difference, v, were an independent variable. To some extent, however, it must vary as stomatal conductance, gs, changes (as is the case for a single leaf). A reduction in gs, and therefore in E, meansmoreheat has to be lost by sensible heat transfer. The-presenceof a leaf boundarylayer resistance to the transfer of heat translates this into an increase of leaf temperatureand of ~ and so the effect of decreased gs on E is moderated. This moderating effect increases as the ratio of boundarylayer resistance to stomatal resistance increases. With a sufficiently high ratio, the proportional reduction in E caused by partial stomatal closure is no greater than the associated proportional reduction in A. Farquharet al (36) discussed the aboveproblemsand defined the conditions that wouldbe necessary for A/E to becomeindependent of stomatal conductance, Pi/Pa and A. The problemis exacerbated in the field, where the aerodynamicresistance of the crop has to be taken into account. If the canopyand leaf boundarylayer

Annual Reviews www.annualreviews.org/aronline CARBONISOTOPE DISCRIMINATION 523 resistances to heat are very large, there is the possibility that a genotypewith a greater stomatal conductancethan another otherwise identical genotype will have a greater value of W(15), despite also having a greater A (36). This morelikely to occur at high temperatures. Onthe other hand, it is less likely to occur whencrops have very small leaf area indexes, as wouldnormally be the case under conditions where stress occurs early, and in crops sownin areas prone to severe, early water stress, because under these conditions the crop is moreclosely "coupled"to the atmosphere,like an isolated plant (15, 66). If the source of variation in A is the capacity for photosynthesis, the effects of boundarylayers are unimportant(15). This appears to be the case for peanuts (62). Therefore at the crop level, identification of the causes underlying differences in A may become important-~differences in conductance having different micrometeorological consequences from differences in photosynthetic capacity. Carbon Isotope Characteristics

Discrimination

and Plant

Growth

Hubicket al (62) found a negative relationship betweendry matter production and A of peanut cultivars grownin field trials. Onthe other hand Condonet al (14) sawa positive relationship betweenyield and/Xfor wheatcultivars in two years that included periods of greater than usual rainfall. The sign of the relatiotlship under well-wateredconditions is difficult to predict. It is clear that any associations betweenA and patterns of carbon partitioning will be important. The relative growth rate, r (sec’l), of a plant depends on the assimilation rate per unit leaf area, A (molC -2 sec-~), and the ratio of t otal plant carbon to leaf area, p (mol C m-2), according to the following identity

(84) r =

IA(1 - ~b~) , P

20.

where l is the photoperiod as a proportion of a day. Masle &Passioura (85) observed that wheat seedlings grewmoreslowly in soil of increased strength than in controls. Masle & Farquhar (84) showed that 19 increased with increasing soil strength. Theyalso foundthat A decreasedwith increasing soil strength. Changingsoil strength thus induced a negative relationship between p and A. Theynoted that a similar, negative, but genetic association between p and A wouldtend to cause a positive relationship betweengrowth rate and A. A negative association between 19 and A has been observed amongwheat and sunflower genotypes during early growth (J. Virgona, personal communication).If v is lowearly in the life of a crop, then a positive association between A and relative growth rate among genotypes will confound the relationship between final Wand A.

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FARQUHARET AL

Genetic Control of Discrimination Genetic studies of W, Pi/p~, and A are in their infancy. Thesetraits are most likely to be polygenic, since any genethat affects either assimilation rate per unit leaf area or stomatal conductancecan have an effect. Despite the considerable genetic and environmental(nutrition, light intensity, etc) effects the individual components A and g, separately, it is likely that the variation in the ratio A/g, and hence in pi/p~ and A, is smaller, because of coordination between A and g (37). The coordination can lead to predictable genotypic differences in P~/Paand A as assessed from gas exchange(62), as well as in A assessed from 6p. The genetic control of A appears to be strong in wheat. Condonet al (14) showedthat genotypic ranking was maintained at different sites and between plants grownin pots and in the field. The broad sense heritabilities [proportion of total variance of A that can be ascribed to genotype, rather than to environment or to interactions between the two (G × E)] ranged between and 90%. From analyses of A in 16 peanut genotypes grown at 10 sites in Queensland,Hubicket al (62) calculated an overall broad sense heritability 81%. With Phaseolus vulgaris in Colombia, it was 71%(23). Hubick et (62; and see earlier discussion in reference 36) examinedthe progeny of cross between Tifton 8, a peanut genotype having a small value of A, and Chico, which has a large value of A. Statistical analyses of measurementsof A and Win the F2 generation gave estimates for the heritability of 53%for A and 34%for W, The phenotypic correlation between Wand A was --0.78. As expected, the A values of F2 plants were highly variable and there were several transgressive segregants with values of A lower than those of Tifton 8. The A values of the F~ generation of the Tifton 8 and Chico cross, while somewhatintermediate between the two parents, were very close to those of Tifton 8 in A and W.Martin &Thorstenson (83) examinedthe F~ plants from a cross betweenLycopersiconpennellii, a drought-tolerant species related to tomato, with tomatoitself, Lycopersiconesculenturn. L. pennellii had a lower A than L. esculentum, and again A of the F1 was intermediate, but closer to the low-A parent. Both sets of data suggest some dominanceof the low-A attribute. Genetic analysis of a polygenic trait like A is obviously difficult, yet considerable progress has recently been made using modemtechniques. Martin et al (82) reported that 70%of the variance for A in a variable tomato population derived from further generations of the above cross was associated with three restriction fragment length polymorphisms(RFLPs)~i.e. genetic markers identifying discrete DNAsequences within the genome. Additive gene action was observed in the three cases, and in one of them, there was also a significant nonadditive component. This kind of work may enable breeders to follow the results of backcrossing material with desirable A into

Annual Reviews www.annualreviews.org/aronline CARBONISOTOPE DISCRIMINATION 525 commercial cultivars. However,in parallel with pursuing research on the genetic control of carbonisotope discriminationby the plant, it is importantto establish what values of A are appropriate in a particular environmentand for a particular species. This requires extensive physiological workat different scales, from the organelle to the canopy, and a muchbetter understanding of the interactions amongplants, canopies, and their microclimates. CONCLUDING

REMARKS

Carbon isotope discrimination has becomea tool to help us understand photosynthesis and its coordination with water use in ecological and physiological studies of C3 species. Future workwill relate these moreto growth characteristics and will differentiate betweeneffects of photosyntheticcapacity and stomatal conductance. The latter may perhaps be studied using observations of isotopic composition of organic oxygen and hydrogen(36). These compositions are affected by the ratio of ambient and intercellular humidities and should therefore reflect changes in the energy budgets of leaves, which are themselves influenced by stomatal conductance. It is possible that measurementsof A in C4 species mayaid in seeking changes in coordination between mesophyll and bundle sheath tissue during photosynthesis, perhaps revealing differences in quantumrequirements. Technological advances in combining gas chromatography and isotope ratio mass spectrometry should facilitate measurementsof carbon isotope discrimination between and within organic compounds,thereby increasing our ability to identify origins of materials and to study the nature of the control of metabolic pathways following photosynthesis. ACKNOWLEDGMENTS

Wethank Drs. Joe Berry and Josette Masle for valuable discussions and commentson this manuscript. APPENDIX

Part 1. Definitions Isotope effects (c~) are here defined as the ratio of carbon isotope ratios in reactant and product (39) A1. whereR~is the 13C/12C molar ratio of reactant and Rp is that of the product. in a first order kinetic reaction, the definition is obvious, i.e.

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FARQUHARET AL ~2 k

A2.

wherek~2 and k~3 are the rate constants for reactions of the respective isotopic substances. Higher-order kinetic reactions including Michaelis-Mentenones (38) can be treated similarly (102), and ~2 and k13 become speudo-first-order rate constants. The isotope effect associated with diffusion is the ratio of the ~2Cand ~3Cdiffusivities. The analogy with Equation AI is the diffusion from a source (reactant) to a sink wherethe "product"is kept at a vanishingly small concentration. In an equilibrium, the "product" is the carbon-containing compound of interest on the right-hand side of an equilibrium reaction. So if the reaction of interest is kl

A3.

A ~B, k_~

where A and B might be COzand HCO~-,for example, then application of this rule yields ~3 A Al~ ¢~ = B13

--

Kl2 K13 ,

A4.

where K~2 is the equilibrium constant, A5. for the ~2Ccompoundsand K~3 is the analogous constant for 13C compounds. Notethat the equilibriumisotope effect, ~x, is the kinetic isotope effect for the forward reaction (a0 divided by that of the reverse reaction (c~_~---i.e.

It is pleasing then that the formsof the isotope effect (t~) for kinetic effects (k~2/k13) and equilibrium effects (KtZ/K13) are superficially similar. Wedenote the discrimination for either effect as ct minusone (39). In most cases

Annual Reviews www.annualreviews.org/aronline CARBONISOTOPE DISCRIMINATION 527 discriminationassociated with a kinetic effect will be positive, but there is no a priori reason whya thermodynamicdiscrimination should be positive. Part 1I. Discrimination in a simple two stage model---diffusion followed by carboxylation The carbon isotope ratio of CO2in air is R~, and in the plant product is Re. In turn Rp must be the sameas the ratio of ~3CO2 assimilation rate, A13, and ~2CO2 assimilation rate, A [no superscript is given here for a variable relating to the major isotope ~2C]--i.e. 13 A

A7.

Re = A

Further, if the isotope effect associated with carboxylation is 1 + b, then we must have gi

--=l+b,

A8.

whereRi is the carbon isotope ratio of the intercellular C02. In turn, Ri is simply found by relating A to g (conductance) and P (total pressure). Thus, A = g(P" - Pl) P

A9.

Thekinetic isotopeeffect for diffusion is the ratio of the diffusivities of lZCO2 and ~3CO2in air. Thus, 1 + a = ~g

AI0.

g13 ’

and so AI 3 =

g(Rap~ - Ripl) (1 + a)P

All.

Substituting Equations A9 and A11 in A7, Rap,~ -i Rip (1 + a)(pa -Pi)"

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FARQUHARET AL

Rearranging, R~ --=(1

Ri Pi

Pa - Pi

+a)

Thus, using the definition of discrimination and Equation A8 a 1 + A Ro (1 + a) Rp

p° - pi + (1 + b) pi Pa Pa

Thus A = a Pa -- Pi + b P__L, Pa Pa whichis Equation8 fromthe maintext. Notethat no assumptionof linearity is madeabout the response of A to p~ in the derivation of this equation. Part 1II. Alternative definitions of discrimination There are other possible definitions of discrimination. For exampleone could write Discrimination*

= 1 - --

This would correspond to (1 - kl3/k 12) for kinetic effects and to (1 Kla/K12) for equilibrium effects. The asterisk superscript is addedto emphasize that the numericalvalues obtained differ from those madeusing Equation 4. On this basis

The numerical differences between this and our chosen definition of discrimination are usually less than 0.5%0. In the case of discrimination by ribulose bisphosphate carboxylase (Rubisco), the two definitions differ by 0.9%0, which is significant. However,formulation of discrimination as A* rather than as (Ra/Rt, - 1), wouldmakederivation of the theory muchmore complicated.This maybe seen by repeating the derivation in Part II ,using a* = 1 - gl3/g and b* = 1 - Rp/Ri. Althoughit mayseem odd to have the abundanceratio of the source, Ra, in the numerator of our chosen definition (Equation A1), we note that RdR~may equally be thought of as SplSa, where S is the molar ratio 12C/13C.

Annual Reviews www.annualreviews.org/aronline CARBONISOTOPE DISCRIMINATION 529 Yet another notation is to use Rp/R~- 1, kl3/k which leads to negative values of discrimination.

12 -

1, and K13/K12

- I,

Part IV. Complications to the use of A = a + (b - a)pi/Pa Farquhar (34) showedthat the appropriate value of Pi in Equation8 is the assimilation-rateweighted value of pi, whereas normal gas exchange gives a conductanceweightedvalue of p;. Thesetwo estimates will differ if there is heterogeneity of stomatal opening(73, 141) and restricted lateral diffusion within the leaf. Greater degrees of heterogeneity will therefore cause smaller best fit values for b. The simplest value of b wouldbe the isotope discrimination factor of Rubiscocarboxylation, taking gaseous COzas the substrate (b3). Roeske O’Leary(119) measured the isotope effect as 1.029, but with respect dissolved CO2,so that the result must be multiplied by the isotope effect of the dissolution of CO2in water (1.0011) makingb3 approximately30%0(36). Guyet al (50) measured the effect directly with respect to the gas monitoring continuing isotopic enrichment of CO2in a reaction vessel and calculated b3 to be -- 29%0using an equation analogous to that for Rayleigh distillation (7, 97). However,Farquhar&Richards (39) suggested that the discrimination in Ca photosynthesis should be less than that in the Rubisco carboxylation, because even in Ca species a portion,/3, of COzfixation is via PEPcarboxylase. With b4 being the net fractionation by PEPcarboxylase with respect to gaseous CO2in equilibrium with HCO~,(-5.7%0; see Table 1) they suggested a net discrimination value of b = (l-~)b3

+ Bb4 = b3 - ~(b~ - b4).

Thedifference (b3 - b4) is - 36%0,so that b is sensitive to the proportion of /3-carboxylation. The latter depends on the amount of aspartate to be formed~unlikely to vary muchbetween plants--and the amount of HCO~formed for pH balance. This latter factor maycontribute to the greater discrimination shownby Ricinus plants grown with NH~-as N source than whenNO~-was the sole source, although the phenomenonwas interpreted in terms of changedstomatal behavior (114). In an unpublished study by Melzer & O’Leary (personal communication), Ca fixation was found to reduce carbon discrimination by no more than 1%oin C3 species. AssumingPi/Pa was ~ 0.7, this means that b could be reduced from b3 by 1.9%o. Other effects are ignored in the simple modelrepresented by Equation 8. Theseinclude the presence of resistance betweenthe intercellular spaces and the sites of carboxylation, and effects of respiratory losses and translocation. Manyof these effects are taken into account in a moredetailed equation (32) for which Equation 8 is a simplification:

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FARQUHAR ET AL

eR__~+ fF* A

=

P. - Ps ab --+ Pa

Ps - Pi a~ + (es Pa

+

Pi - P~ a])~+ Pa

Pc Pa

k Pa

,A12.

whereps is the p(CO2) at the leaf surface, pc is the equivalent p(CO2) at the sites of carboxylation,ab is the fractionation occurringduring diffusion in the boundary layer (2.9%o), es is the fractionation occurring as CO2enters solution [1.1%oat 25°C;(149)] at is the fractionation due to diffusion in water [0.7%0; (98)], e and f are fractionations with respect to average carbon composition associated with "dark" respiration (Rd) and photorespiration, respectively, k is the carboxylation efficiency, and F* is the CO2compensation point in the absence of Rd (32). Equation 8 overestimates discrimination compared to Equation A12by ee d

d = [rb(a

-- ab) +

AP

rw(b - es - at)]

+

Pa

A13. Pa

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